NMR-Based Method for Intramolecular 13 C Distribution at Natural Abundance Adapted to Small Amounts of Glucose.

Quantitative nuclear magnetic resonance (NMR) for isotopic measurements, known as irm-NMR (isotope ratio measured by NMR), is well suited for the quantitation of 13 C-isotopomers in position-specific isotope analysis and thus for measuring the carbon isotope composition (δ 13 C, mUr) in C-atom positions. Irm-NMR has already been used with glucose after derivatization to study sugar metabolism in plants. However, up to now, irm-NMR has exploited a "single-pulse" sequence and requires a relatively large amount of material and long experimental time, precluding many applications with biological tissues or extracts. To reduce the required amount of sample, we investigated the use of 2D-NMR analysis. We adapted and optimized the NMR sequence so as to be able to analyze a small amount (10 mg) of a glucose derivative (diacetonide glucofuranose, DAGF) with a precision better than 1 mUr at each C-atom position. We also set up a method to correct raw data and express 13 C abundance on the usual δ 13 C scale (δ-scale). In fact, due to the distortion associated with polarization transfer and spin manipulation during 2D-NMR analyses, raw 13 C abundance is found to be on an unusual scale. This was compensated for by a correction factor obtained via comparative analysis of a reference material (commercial DAGF) using both previous (single-pulse) and new (2D) sequences. Glucose from different biological origins (CO 2 assimilation metabolisms of plants, namely, C 3 , C 4 , and CAM) was analyzed with the two sequences and compared. Validation criteria such as selectivity, limit of quantification, precision, trueness, and robustness are discussed, including in the framework of green analytical chemistry.